U.S. patent application number 14/766558 was filed with the patent office on 2015-12-31 for genetically engineered bacterium for treatment of breast cancer, method for constructing the bacterium, and applications thereof.
This patent application is currently assigned to Nanjing Sinogen Biotech & Pharmaceutical Inc.. The applicant listed for this patent is NANJING SINOGEN BIOTECH & PHARMACEUTICAL INC.. Invention is credited to Fanghong Li, Xiaoxi Li, Yan Lin, Pengli Yu, Allan Zhao, Sujin Zhou.
Application Number | 20150376593 14/766558 |
Document ID | / |
Family ID | 48544949 |
Filed Date | 2015-12-31 |
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United States Patent
Application |
20150376593 |
Kind Code |
A1 |
Lin; Yan ; et al. |
December 31, 2015 |
GENETICALLY ENGINEERED BACTERIUM FOR TREATMENT OF BREAST CANCER,
METHOD FOR CONSTRUCTING THE BACTERIUM, AND APPLICATIONS THEREOF
Abstract
The current invention discloses a genetically engineered
bacterium used for the treatment of breast cancer. The said
bacterium is attenuated Salmonella typhimurium VNP20009 with cloned
L-methioninase gene. The method for constructing this genetically
engineered bacterium and the application thereof are also disclosed
herein. In the current invention, our biologic drug for the
treatment of breast cancer is a type of safe, non-toxic new drug
with anti-tumor activity. It can highly express methioninase
through recombinant DNA technology using attenuated Salmonella
typhimurium VNP20009 as a carrier, which has a strong anti-tumor
activity and can meet the needs. The preparation method is simple
and easy to operate, showing good application prospect.
Inventors: |
Lin; Yan; (Nanjing, Jiangsu,
CN) ; Zhou; Sujin; (Nanjing, Jiangsu, CN) ;
Zhao; Allan; (Nanjing, Jiangsu, CN) ; Li; Xiaoxi;
(Nanjing, Jiangsu, CN) ; Yu; Pengli; (Nanjing,
Jiangsu, CN) ; Li; Fanghong; (Nanjing, Jiangsu,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NANJING SINOGEN BIOTECH & PHARMACEUTICAL INC. |
Nanjing, Jiangsu |
|
CN |
|
|
Assignee: |
Nanjing Sinogen Biotech &
Pharmaceutical Inc.
Nanjing, Jiangsu
CN
|
Family ID: |
48544949 |
Appl. No.: |
14/766558 |
Filed: |
February 27, 2014 |
PCT Filed: |
February 27, 2014 |
PCT NO: |
PCT/CN2014/072652 |
371 Date: |
August 7, 2015 |
Current U.S.
Class: |
424/93.2 ;
435/471 |
Current CPC
Class: |
C07K 14/195 20130101;
C12Y 404/01011 20130101; Y02A 50/481 20180101; A61K 48/005
20130101; A61K 38/00 20130101; C12N 9/88 20130101; C12N 15/74
20130101; Y02A 50/30 20180101; A61P 35/00 20180101 |
International
Class: |
C12N 9/88 20060101
C12N009/88; C12N 15/74 20060101 C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2013 |
CN |
201310062253.7 |
Claims
1. An application of a genetically engineered bacterium for the
treatment of breast cancer, wherein the genetically engineered
bacterium is attenuated Salmonella typhimurium VNP20009 with a
cloned L-methioninase gene.
2. A method for constructing the genetically engineered bacterium
for breast cancer according to claim 1, wherein the L-methioninase
gene is subcloned into pUC57 plasmid, and then subcloned into
pSVSPORT plasmid through Kpn I and Hind III restriction sites to
obtain pSVSPORT-L-methioninase expression plasmid, which then is
transformed via electropolaration into attenuated Salmonella
typhimurium VNP20009, to obtain the genetically engineered
bacterium.
3. The method for constructing the genetically engineered bacterium
for the treatment of breast cancer according to claim 2, wherein
conditions for the electroporation are as follows: voltage 2400 V,
resistor 400 .OMEGA., capacitor 25 .mu.F, time constant 4 ms.
4. The application of genetically engineered bacterium according to
claim 1 in preparing drugs for treatment of breast cancer.
Description
TECHNICAL FIELD
[0001] The current invention relates to drugs for the treatment of
cancers, in particular, to the construction and applications of the
genetically engineered bacterium in preparing the drugs for the
treatment of breast cancer.
BACKGROUND OF THE INVENTION
[0002] Breast cancer is one of the common malignancies of women,
and its incidence is in the first place among female cancer
patients. Currently the primary treatment procedures for breast
cancer include surgery, radiotherapy, chemotherapy, and hormone
therapy, etc. Chemotherapy is always playing an important role in
the comprehensive treatment of breast cancer due to the sensitivity
of breast cancer to anti-cancer drugs Traditional drugs for
chemotherapy in breast cancer mainly include doxorubicin,
cyclophosphamide, 5-fluorouracil, etc. Although these drugs have
been widely used in the treatment of breast cancer, their
therapeutic efficacy is limited due to the toxicity and resistance
of patients. In recent years, targeted therapy is used clinically,
with the primary drugs like Herceptin, tyrosine kinase inhibitor,
lapatinib, pertuzumab monoclonal antibody, bevacizumab,
flavopiridol, etc. However, due to patient's tolerance, physical
properties of drugs like instability and solubility and the
targets, targeted therapy only applies to a subgroup of patients.
With the advances in bacterial- and viral-based gene therapy and
genetic engineering technology, mounting studies have focused on
bacterial treatment of tumors since the middle 1990s. Results have
shown that Salmonella typhimurium can inhibit the growth of tumor
cells in mice in a targeted and efficient manner.
[0003] Salmonella is a group of Gram-negative, invasive
intracellular facultative anaerobes parasitized in human and animal
intestinal tracts. VNP20009 is an attenuated Salmonella typhimurium
strain with the deletion of msb B and pur I genes. It is
genetically stable and sensitive to antibiotics. The msb B protein
is necessary for the lipid acylation to endotoxin, and the lipid
acylation at A-terminal cannot be achieved when deleted, lowering
the toxicity. The pur I protein is involved in purine metabolism,
deletion of this gene leads to dependence of exogenous adenine when
culturing the bacteria. These gene manipulations in VNP20009 also
lower the production of tumor necrosis factor (TNF), thereby
reducing the inflammatory response. Consequently, the low
pathogenicity improves the safety of its clinical usage. VNP20009
has been widely used in cancer research, which can influence the
growth of a variety of solid tumor models of mice, including
melanoma, lung cancer, colon cancer, breast cancer, renal cancer
and prostate cancer. VNP20009, as a vector of gene therapy, has the
ability to accumulate in the tumor site in a highly targeted
fashion. Researchers have found in the mouse models carrying a
variety of solid tumors that the quantity of VNP20009 in tumors is
200-1000 times as high as that in non-cancerous major organs, such
as the liver. It uses a more complex set of mechanisms to target
tumors. VNP20009 can preferentially accumulate and multiply under
the hypoxic and necrotic conditions in the tumor tissue. At the
same time, the bacteria multiply significantly faster in the tumor
tissues than in the normal tissues, making it possible for the
attenuated Salmonella to be a new type of anti-tumor agent and the
vector of targeted gene therapy. Potential mechanisms for the
effect of a slow tumor growth by VNP20009 may include the follows:
1) Breakdown of nutrients necessary for tumor growth by the
bacteria, e.g., the enzymes produced by bacteria such as
asparaginase, can deplete essential amino acids for tumor growth;
2) Stimulation of local toxin secretion or tumor necrosis factor a
to tumor microenvironment can negatively influence the tumor
angiogenesis. In addition, the non-specific inflammatory reaction
at the bacterial growth site can activate anti-tumor T cells.
Studies have shown that although attenuated Salmonella VNP2009 is
an ideal carrier for gene therapy which can be applied safely with
high allowable dose, its application independently has no strong
anti-tumor effect and a further combination with other drugs is
needed.
[0004] Tumor cells require adequate nutrition in order to maintain
its high rate of reproduction. In addition to carbohydrates, the
need for methionine (Met), glutamine, and arginine is particularly
high. Previous studies have established that Met-dependency is a
common feature of most tumor cells, such as breast cancer, lung
cancer, colon cancer, kidney cancer, bladder cancer, melanoma,
glioma, etc. High Met-dependency does not exist in normal cells.
Both in vivo and in vitro experiments have confirmed that dietary
intervention with methionine deficiency can delay the proliferation
of tumor cells. However, long-term deficiency of Met can cause
malnutrition, metabolic disorders, and aggravate tumor growth due
to a long-term DNA hypomethylation. Thus, by specifically degrading
Met to methylselenol, a-ketobutyrate and ammonia through
L-methioninase and lowering the level of methionine in vivo, we
will be able to effectively inhibit the growth of tumor cells or
even degrade them. Experiments in animal models have confirmed that
intraperitoneal injection of methioninase can inhibit the growth of
Yoshida sarcoma and lung tumor in nude mice. In previous clinical
trials, four patients with breast cancer, lung cancer, kidney
cancer and lymphoma received methioninase injection once every 24
h. Methioninase could significantly reduce the methionine content
in plasma. However, since methioninase is not natively expressed in
mammalians, exogenous administration often causes the immunological
response.
SUMMARY OF THE INVENTION
[0005] The first technical know-how in the current invention is to
provide a genetically engineered bacterium for effective treatment
of breast cancer. The strain is safe and non-toxic with anti-tumor
activity and it can meet the clinical needs.
[0006] The second technical know-how in the current invention is to
provide the method for constructing the above genetically
engineered bacterium.
[0007] The final technical know-how in the current invention is to
provide the application of the above genetically engineered
bacterium.
[0008] To reach such goal, the current invention deployed the
technical schemes as follows:
[0009] A genetically engineered bacterium for the treatment of
breast cancer, and the said genetically engineered bacterium is
attenuated Salmonella typhimurium VNP20009 with cloned
L-methioninase gene.
[0010] Wherein the said VNP20009 contains pSVSPORT plasmid and the
said L-methioninase gene is cloned on pSVSPORT plasmid.
[0011] The method for the construction of the genetically
engineered bacterium for the treatment of breast cancer is as
follows: the L-methioninase gene is subcloned into pUC57 plasmid,
and then subcloned into pSVSPORT plasmid through Kpn I and Hind III
restriction sites to obtain pSVSPORT-L-methioninase expression
plasmid, which then is transformed into attenuated Salmonella
typhimurium VNP20009, to obtain the genetically engineered
bacterium.
[0012] Wherein the said electroporation condition is as follows:
voltage 2400 V, resistor 400 .OMEGA., capacitor 25 .mu.F, time
constant 4 ms.
[0013] The application of above genetically engineered bacterium in
preparing drugs for treatment of breast cancer.
[0014] The current invention provides a genetically engineered
tumor-targeting bacterium. It has tumor targeting and can
continuously express L-methioninase in tumor tissues, which then
consume methionine and a series of other nutrients, and depletes
the tumor cells of nutrition, causing slow growth. Besides, the
strain possibly activates caspase-3 apoptosis signaling pathway,
leading to the death of the host tumor cells. Therefore, it can be
used as the drug for the treatment of breast cancer.
[0015] Beneficial effects: compared with prior technology, our drug
used for the treatment of breast cancer is a new, safe, non-toxic
biological drug with anti-tumor activity, which can highly express
methioninase through recombinant DNA technology using attenuated
Salmonella typhimurium VNP20009 as a carrier. It can meet the needs
with a strong anti-tumor activity. The preparation method is simple
and easy to operate, showing good application prospect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows 1% agarose gel electrophoresis by plasmid
pSVSPORT-L-methioninase following restriction enzyme digestion.
[0017] FIG. 2 shows methioninase expression identification by
Western blot.
[0018] FIG. 3 shows the influence of Salmonella injection on the
body weight of nude mice.
[0019] FIG. 4 shows the results of Salmonella distribution
following intratumoral injection in nude mice.
[0020] FIG. 5 shows the tumor size 2 weeks after administration of
Salmonella.
[0021] FIG. 6 shows the tumor weight 2 weeks after administration
of Salmonella.
[0022] FIG. 7 shows the tumor size 2 weeks after administration of
L-methioninase.
[0023] FIG. 8 shows the tumor weight 2 weeks after administration
of L-methioninase.
DETAILED DESCRIPTION OF THE EMBODIMENT
[0024] The invention is described herein in connection with
drawings and certain specific embodiments. However, to the extent
that the following detailed description is specific to a particular
embodiment or a particular use, this is intended to be illustrative
only and is not to be construed as limiting the scope of the
invention.
Example 1: Construction of Genetically Engineered Bacterium
(1) Construction of Plasmids Expressing L-Methioninase Gene
[0025] Firstly, the L-methioninase (GenBank: L43133.1) is
synthesized and subcloned into pUC57 plasmid (GenScript
Corporation), then subcloned into plasmid pSVSPORT (Invitrogen)
through Kpn I and Hind III restriction sites, to get
pSVSPORT-L-methioninase expressing plasmid. The specific procedures
are as follows:
[0026] Double enzyme digestion of plasmid pSVSPORT with Kpn I and
Hind III: 2 .mu.g plasmid DNA, 3 .mu.L 10.times. buffer, 1.5 .mu.L
Kpn I, 1.5 .mu.L Hind III. Add ddH.sub.2O to 30 .mu.L and incubate
at 37.degree. C. for 3 h, and then separate the digests by 1%
agarose gel electrophoresis, to cut out DNA bands with the size of
4.1 kb, and then purify DNA using the gel recovery and purification
kit.
[0027] The DNA fragments in L-methioninase coding region obtained
by gene synthesis are subcloned into plasmid pUC57 (GenScript
Corporation). Perform restriction digests as follows: 3 .mu.g
plasmid DNA, 3 .mu.L 10.times. buffer, 1.5 .mu.L Kpn I, 1.5 .mu.L
Hind III. Add ddH.sub.2O to 30 .mu.L and incubate at 37.degree. C.
for 3 h. Then separate the digests by 1% agarose gel
electrophoresis. We cut out DNA bands with the size of 1.2 kb, and
then purify DNA using a gel recovery and purification kit.
[0028] The pSVSPORT (Kpn I/Hind III) is ligated to DNA fragment of
L-methioninase coding region (Kpn I/Hind III). Add 2 .mu.L vector,
6 .mu.L inserted fragment, 1 .mu.L T4 DNA ligase in the ligation
reaction, and incubate at 16.degree. C. for 16 h.
[0029] The ligation product is transformed to competent cells of
E.coli DH5.alpha. (Takara). Use one tube 50 .mu.L of DH5.alpha.
competent cells and place on ice until thawing. Add 5 .mu.L of the
above ligation product to the DH5.alpha. and mix them gently, and
then incubate on ice for 30 min; after heat shock at 42.degree. C.
for 60 s, cold shock on ice for 2 min; add 500 .mu.L of LB without
antibiotic and culture at 37.degree. C. with shaking for 1 h; spin
tube at 4000 rpm for 5 min; remove all but 100 .mu.L of LB and
resuspend pellet with pipette tip. Place suspensions on LB plate
containing ampicillin, and then incubate at 37.degree. C. for 16
h.
[0030] When clones grow out, pick up the monoclonal colonies into 3
mL LB containing ampicillin, culture at 37.degree. C. with shaking
for 1 h. Extract the plasmid DNA from cultures and identify by Kpn
I and Hind III restriction analysis. DNA bands of 4.1 and 1.2 kb
are measured in positive clones, as shown in FIG. 1. Then the
positive clone is sent for sequencing to confirm the identity of
the insert fragment.
(2) Construct VNP20009-L-Methioninase Strain
[0031] The plasmid pSVSPORT and pSVSPORT-L-methioninase are
electroporate into VNP20009 strain (YS1646), named VNP20009-V and
VNP20009-M respectively. The specific construction procedures are
as follows:
[0032] Place competent bacteria VNP20009 on ice. After thawing,
transfer it to a pre-cooled electroporation cuvette and add 24
plasmid, slightly mix them, then incubate on ice for 1 min. Put the
cuvette into electroporation apparatus seted to 2400 V, 400
.OMEGA., 25 .mu.F and 4 ms. After pulse, immediately add 1 mL SOC
medium to the cuvette and mix gently. Culture at 37.degree. C. with
shaking for 1 h, centrifuge at 4000 rpm for 5 min and remove all
but 100 .mu.L of LB and resuspend pellet with pipette tip. Plate
the electroporation mixture on LB plate containing ampicillin, and
then incubate at 37.degree. C. for 16 h. After VNP20009-V and
VNP20009-M are cultured with LB, extract the plasmid and
identification by restriction digestion.
[0033] Extract proteins from 1.times.10.sup.8 Salmonella and
separate by 10% SDS-PAGE electrophoresis, transfer to PVDF
membranes in an ice bath. The membranes are blocked by incubation
in BSA at room temperature for 1 h. After three 5-min washes in
TBST, the membranes are incubated at 4.degree. C. overnight with
rabbit antibody against L-methioninase (1:1000). After three 5-min
washes in TBST, the membranes are incubated with horseradish
peroxide-conjugated anti-rabbit secondary antibodies (1:10000) for
1 hr at room temperature. After three 5-min washes in TBST, the
protein bands are visualized using enhanced chemiluminescence (ECL)
reagents. The results are shown in FIG. 2. There is a specific band
at about 43 kD molecular weight, suggesting compared with that of
VNP20009 and VNP20009-V, L-methioninase expression of VNP20009-M is
significantly increased.
Example 2: The Anti-Tumor Effect of VNP20009-L-Methioninase
Strain
[0034] 1. Culture breast cancer cell MDA-MB-231 using MEM medium
containing 10% fetal bovine serum and inoculate 2.times.10.sup.6
cells on the right armpit of nude mice. Observe the state of mice
every 2 to 3 days and measure the tumor size using a vernier
caliper (volume=0.52 .times.length .times.width.sup.2). When the
tumor size reaches 0.1.about.0.2 cm.sup.3 , tumor-bearing mice are
randomized: PBS, VNP20009-V and VNP20009-M groups.
[0035] 2. Culture VNP20009-V and VNP20009-M with LB-O. When
OD.apprxeq.0.6, collect the thallus and re-suspend it in PBS. Mice
are administered by intratumoral injection at a dose of
2.times.10.sup.6CFU each, while the control group are administered
with the same volume of PBS. After administration, observe the
activities, eating Patterns and body weight of nude mice, results
are shown in FIG. 3. After bacterial injection, the body weight of
mice is not affected; moreover, the feeding and feces of nude mice
have no abnormalities, indicating that VNP20009-V and VNP20009-M
have no obvious toxicity to nude mice.
[0036] 3. After administration, on day 2, 12, 20, take major
tissues of nude mice, to grind and homogenize with PBS and culture
them on LB plates overnight after gradient dilution. Results are
shown in FIG. 4--the quantitative colony count results of tissue
homogenate. After two day of intratumoral bacteria injection, the
bacteria count in the tumor tissue is 3.times.10.sup.7 CFU/g, while
no bacteria is detected in liver, kidney, etc. Twelve days later,
the count of bacteria in the tumor tissue is 6.3.times.10.sup.7
CFU/g, while that in the liver is 1.5.times.10.sup.5 CFU/g, to
reach a ratio about 400:1. Twenty days later, the ratio of bacteria
between the tumor tissue and other tissues is about
4000:1.about.35000:1, indicating that VNP20009 has a well targeting
ability to this kind of breast tumor.
[0037] 4. Measure the length and width of the tumor every 2-3 days,
calculate the tumor volume and plot the tumor volume curve of nude
mice. Two weeks after administration, there is a significant
difference in the tumor size between the control and experiment
group. Randomly take three mice from each group, strip the tumor of
the nude mice, weigh it and take photos. The results are shown in
FIG. 5 and FIG. 6, after administration of Salmonella VNP20009-M,
the tumor grows slowly, the tumor volume and weight is about 1/2 of
that in the PBS and VNP20009-V group, but there is no significant
difference between VNP20009-V and PBS group, suggesting that
VNP20009 with high expression of L-methioninase has significant
inhibitory effect on the tumors of breast cancer.
[0038] 5. The procedures are the same as those in 1. Tumor-bearing
nude mice are divided into three groups and administered with PBS,
L-methioninase 1 ng/mouse, L-methioninase 100 ng/mouse by
intratumoral injection. Two weeks later, tumors are stripped,
weighed and photographed. Results are shown in FIGS. 7,8. There is
no significant difference in tumor size and weight among the three
groups. The L-methioninase level in L-methioninase ing/mouse is
equivalent to that contained in 2.times.10.sup.6 CFU VNP20009-M.
Thus, the administration of equal or even 100-fold dose of
L-methioninase shows no significant anti-tumor effects. This
indicates that with the L-methioninase depletion or degradation, a
single administration does not function, while the continuous
high-expression of L-methioninase using VNP20009 as the carrier can
make up this drawback, showing significant anti-tumor effects.
* * * * *